Polyamines are intimately involved in cell growth, cellular response to stress and transformation processes. Polyamine metabolism changes significantly in cells at different stages of cell growth, in cells before and after a specific stress, and in cells before and after transformation from a normal to a neoplastic phenotype. These changes in intracellular polyamine metabolism result in measurable differences in the total amounts of specific polyamines and their metabolites in a given cell as well as specific activities of many enzymes of the polyamine pathway. The function of polyamines in these processes remains unclear. The unifying hypothesis of this proposal is simply stated: changes in intracellular compartmentation of polyamine metabolites play a key role in both the control of polyamine metabolism and polyamine function. It is the relative amounts of polyamine metabolites in a specific compartment which is relevant to these processes. The proposed research will test this hypothesis using nuclear magnetic resonance spectroscopy (NMR/MRS) to non-invasively examine polyamine metabolism in intact cells under well-defined perfusion conditions. By growing cells in defined media, or using specific inhibitors of the polyamine pathway, normal polyamines can be replaced with polyamines labelled with NMR-visible isotopes. This will permit careful characterization of intracellular polyamine pools as defined by specific NMR parameters. Relevant measurements include chemical shifts, signal intensities, and relaxation times for each detectable metabolite, and given appropriate kinetics, reaction rates through specific pathways. Using a variety of labelled compounds and experimental strategies, NMR parameters of intracellular polyamine pools will be determined in a single cell line during exponential growth and at confluence. The same cell line will be further examined while in exponential growth before, during, and after heat shock. Used in conjunction with standard biochemical assays, NMR spectroscopic data obtained from intact cells will provide unique insight into intracellular polyamine metabolism and function. The labelling strategies and methods developed herein will be widely applicable to the study of polyamine metabolism in other cells and tissues; the potential to extend these techniques to intact animals exists. Interference with polyamine metabolism and function has proven a valuable approach to the treatment of neoplasms and other diseases. Further characterization of polyamine metabolism in the intact cell will likely suggest novel therapies as well as further refinement to current approaches.